Abstract: The discovery of RNA interference (RNAi) has impacted the way we understand gene regulation in a major way. Owing to its unique ability to down regulate a specific gene, RNAi has been widely used for biological research and applied for therapeutic purposes. Despite the differences in their origin, both small interfering RNA (siRNA) and micro RNA (miRNA) are processed by the same protein machineries of the RNAi pathway in human. The main components of the RNA silencing proteins include Dicer, TRBP and Argonaute (AGO), which make up the RNA induced silencing complex (RISC). Although the roles of individual components are delineated, the interplay between RNA and protein components and the mechanism involved in strand selection and separation has not been elucidated, mainly due to the lack of measurement tools available. Moreover, recent studies report various off-target effects which result in unintended gene silencing or extremely low efficiency of silencing even when the RNA substrates are optimized for its maximal effect. We propose to develop series of single molecule and single cell measurement to dissect the RNAi pathway step by step. Our proposal entails four stages of tests as detailed below. First stage is pre-scanning of RNA by TRBP. It is based on our recent discovery that TRBP slides on double stranded RNA (dsRNA) in ATP independent manner. TRBP sliding displays substrate dependens which implies its potential role in pre-scanning RNAi substrates. We will subject various pre-siRNA and pre-miRNA to test the selectivity of TRBP sliding. Second test is Dicer-TRBP mediated cleavage in real time. Using our established single molecule platform, we expect to measure the cleavage rate of various pre-siRNA and pre- miRNA substrates as demonstrated by our preliminary data. This will be a direct measure of the dicing efficiency. Third stage involves Dicer-TRBP-AGO, triplex induced unwinding and strand selection, where AGO will be pulled down from mammalian cell extract. The result from this test will unfold the previously hidden steps and players responsible for strand separation and selection with high precision. Fourth step employs single molecule fluorescence in situ hybridization (smFISH) to directly measure the silencing efficacy of a given RNAi substrate. The smFISH enables one to count the number of individual mRNAs in a single cell and this will be serve as a direct indicator of silencing. The smFISH data will be complemented by immunofluorescence to assess the silencing effect at the protein level. We hypothesize that the efficiency at each step of RNAi pathway to be examined under stage 1-3 will be correlated with how well the RNAi substrate will silence the target mRNA, making these tests a prediction/optimization tool for RNAi application. When engineered into a device, such technique can be adopted by research laboratories and clinical practices for designing siRNA for high silencing potency. In summary, our proposal involves developing novel single molecule platforms which allow quantitative and stepwise analysis of RNAi pathway and opens a new way of assessing and predicting siRNA efficacy. Public Health Relevance: This proposal seeks to dissect the RNA interference pathway by using series of single molecule platforms with the goal of developing a prediction tool for siRNA design. This tool will enable assessment and optimization of siRNA substrate to be applied for therapeutic purposes. We will test the RNAi treatment on melanoma cells for down regulating Bcl-2 and C-myc oncogenes.